Methods and compositions utilizing quinazolinones

ABSTRACT

Quinazolinones of formulae (a, b, c and d) are disclosed. They are useful for treating cellular proliferative diseases and disorders associated with KSP kinesin activity.

FIELD OF THE INVENTION

This invention relates to quinazolinone derivatives, which areinhibitors of the mitotic kinesin KSP and are useful in the treatment ofcellular proliferative diseases, for example cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation.

BACKGROUND OF THE INVENTION

Interest in the medicinal chemistry of quinazoline derivatives wasstimulated in the, early 1950's with the elucidation of the structure ofa quinazoline alkaloid,3-[β-keto-gamma-(3-hydroxy-2-piperidyl)-propyl]4-quinazolone, from anAsian plant known for its antimalarial properties. In a quest to findadditional antimalarial agents, various substituted quinazolines havebeen synthesized. Of particular import was the synthesis of thederivative 2-methyl-3-o-tolyl-4-(3H)-quinazolinone. This compound, knownby the name methaqualone, though ineffective against protozoa, was foundto be a potent hypnotic.

Since the introduction of methaqualone and its discovery as a hypnotic,the pharmacological activity of quinazolinones and related compounds hasbeen investigated. Quinazolinones and derivatives thereof are now knownto have a wide variety of biological properties including hypnotic,sedative, analgesic, anticonvulsant, antitussive and anti-inflammatoryactivities.

Quinazolinone derivatives for which specific biological uses have beendescribed include U.S. Pat. No. 5,147,875 describing 2-(substitutedphenyl)-4-oxo quinazolines with bronchodilator activity. U.S. Pat. Nos.3,723,432, 3,740,442, and 3,925,548 describe a class of1-substituted-4-aryl-2(1H)-quinazolinone derivatives useful asanti-inflammatory agents. European patent publication EP 0 056 637 B1claims a class of 4(3H)-quinazolinone derivatives for the treatment ofhypertension. European patent publication EP 0 884 319 A1 describespharmaceutical compositions of quinazolin-4-one derivatives used totreat neurodegenerative, psychotropic, and drug and alcohol inducedcentral and peripheral nervous system disorders. Quinazolinones areamong a growing number of therapeutic agents used to treat cellproliferative disorders, including cancer. For example, PCT WO 96/06616describes a pharmaceutical composition containing a quinazolinonederivative to inhibit vascular smooth cell proliferation. PCT WO96/19224 uses this same quinazolinone derivative to inhibit mesengialcell proliferation. U.S. Pat. Nos. 4,981,856, 5,081,124 and 5,280,027describe the use of quinazolinone derivatives to inhibit thymidylatesynthase, the enzyme that catalyzes the methylation of deoxyuridinemonophosphate to produce thymidine monophosphate which is required forDNA synthesis. U.S. Pat. Nos. 5,747,498 and 5,773,476 describequinazolinone derivatives used to treat cancers characterized byover-activity or inappropriate activity of tyrosine receptor kinases.U.S. Pat. No. 5,037,829 claims (1H-azol-1-ylmethyl) substitutedquinazoline compositions to treat carcinomas which occur in epithelialcells. PCT WO 98/34613 describes a composition containing aquinazolinone derivative useful for attenuating neovascularization andfor treating malignancies. U.S. Pat. No. 5,187,167 describespharmaceutical compositions comprising quinazolin-4-one derivativeswhich possess anti-tumor activity.

Other therapeutic agents used to treat cancer include the taxanes andvinca alkaloids. Taxanes and vinca alkaloids act on microtubules, whichare present in a variety of cellular structures. Microtubules are theprimary structural element of the mitotic spindle. The mitotic spindleis responsible for distribution of replicate copies of the genome toeach of the two daughter cells that result from cell division. It ispresumed that disruption of the mitotic spindle by these drugs resultsin inhibition of cancer cell division, and induction of cancer celldeath. However, microtubules form other types of cellular structures,including tracks for intracellular transport in nerve processes. Becausethese agents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins that have been identified is KSP. KSP belongsto an evolutionarily conserved kinesin subfamily of plus end-directedmicrotubule motors that assemble into bipolar homotetramers consistingof antiparallel homodimers. During mitosis KSP associates withmicrotubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339414 (1996); Blangy, et al.,J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426), and a fragment of theKSP gene (TRIP5) has been described [Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number L40372]. Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

Mitotic kinesins are attractive targets for the discovery anddevelopment of novel mitotic chemotherapeutics. Accordingly, it is anobject of the present invention to provide methods and compositionsuseful in the inhibition of KSP, a mitotic kinesin.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compositions and methods that can be used to treat diseases ofproliferating cells. The compositions are KSP inhibitors, particularlyhuman KSP inhibitors.

In one aspect, the invention relates to methods for treating cellularproliferative diseases, for treating disorders associated with KSPkinesin activity, and for inhibiting KSP kinesin. The methods employcompounds chosen from the group consisting of:

wherein:

-   -   R₁ is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₂ and R₂′ are independently chosen from hydrogen, alkyl,        oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,        substituted alkyl, substituted aryl, substituted alkylaryl,        substituted heteroaryl, and substituted alkylheteroaryl; or R₂        and R₂′ taken together form a 3- to 7-membered ring;    -   R₃ is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted        oxaalkylaryl, oxaalkyl heteroaryl, substituted        oxaalkylheteroaryl, R₁₅O— and R₁₅—NH—;    -   R_(3′) is chosen from hydrogen, alkyl, aryl, alkylaryl,        heteroaryl, alkylheteroaryl, substituted alkyl, substituted        aryl, substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl and R₁₅—NH—;    -   R_(3″) is chosen from alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₄ is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl, and R₁₆-alkylene-;    -   R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, alkyl,        alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino,        alkylsulfonyl, alkylsulfonamido, sulfonamidoalkyl,        sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,        aminocarbonyl, aryl and heretoaryl;    -   R₁₅ is chosen from alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₁₆ is chosen from alkoxy, amino, alkylamino, dialkylamino,        N-heterocyclyl and substituted N-heterocyclyl.

Diseases and disorders that respond to therapy with compounds of theinvention include cancer, hyperplasia, restenosis, cardiac hypertrophy,immune disorders and inflammation; especially cancer, hyperplasia,restenosis, and cardiac hypertrophy; particularly cancer.

In another aspect, the invention relates to compounds useful ininhibiting KSP kinesin. The compounds have the structures shown above.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to a KSP kinesin, for examplecompounds that will displace or compete with the binding of thecompositions of the invention. The methods comprise combining a labeledcompound of the invention, a KSP kinesin, and at least one candidateagent and determining the binding of the candidate bioactive agent tothe KSP kinesin.

In a further aspect, the invention provides methods of screening formodulators of KSP kinesin activity. The methods comprise combining acomposition of the invention, a KSP kinesin, and at least one candidateagent and determining the effect of the candidate bioactive agent on theKSP kinesin activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a generic synthetic scheme to make compositions of theinvention.

FIG. 2 depicts a synthetic route for the synthesis of quinazolinone KSPinhibitors.

FIG. 3 depicts representative chemical structures of quinazolinone KSPinhibitors.

FIG. 4 depicts a synthetic route to substantially pure singleenantiomers.

FIG. 5 depicts synthetic routes to sulfonamides (5a), carbamates (5b),ureas (5c) and amines (5d).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a class of novel compounds, basedon a core quinazolinone structure, that are modulators of mitotickinesins. By inhibiting or modulating mitotic kinesins, but not otherkinesins (e.g., transport kinesins), specific inhibition of cellularproliferation is accomplished. Thus, the present invention capitalizeson the finding that perturbation of mitotic kinesin function causesmalformation or dysfunction of mitotic spindles, frequently resulting incell cycle arrest and cell death. The methods of inhibiting a human KSPkinesin comprise contacting an inhibitor of the invention with a KSPkinesin, particularly human KSP kinesins, including fragments andvariants of KSP. The inhibition can be of the ATP hydrolysis activity ofthe KSP kinesin and/or the mitotic spindle formation activity, such thatthe mitotic spindles are disrupted. Meiotic spindles may also bedisrupted.

An object of the present invention is to develop inhibitors andmodulators of mitotic kinesins, in particular KSP, for the treatment ofdisorders associated with cell proliferation. Traditionally, dramaticimprovements in the treatment of cancer, one type of cell proliferativedisorder, have been associated with identification of therapeutic agentsacting through novel mechanisms. Examples of this include not only thetaxane class of agents that appear to act on microtubule formation, butalso the camptothecin class of topoisomerase I inhibitors. Thecompositions and methods described herein can differ in theirselectivity and are preferably used to treat diseases of proliferatingcells, including, but not limited to cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.

Accordingly, the present invention relates to methods employingquinazolinone amides of formula 1a:

quinazolinone sulfonamides of formula 1b

and quinazolinone amines of formulae 1c and 1d

wherein:

-   -   R₁ is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₂ and R₂′ are independently chosen from hydrogen, alkyl,        oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,        substituted alkyl, substituted aryl, substituted alkylaryl,        substituted heteroaryl, and substituted alkylheteroaryl; or R₂        and R₂′ taken together form a 3- to 7-membered ring;    -   R₃ is chosen from hydrogen, allyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted        oxaalkylaryl, oxaalkylheteroaryl, substituted        oxaalkylheteroaryl, R₁₅O— and R₁₅—NH—;    -   R_(3′) is chosen from hydrogen, alkyl, aryl, alkylaryl,        heteroaryl, alkylheteroaryl, substituted alkyl, substituted        aryl, substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl, and R₁₅—NH—;    -   R_(3″) is chosen from alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₄ is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted allyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, substituted        alkylheteroaryl, and R₁₆-alkylene-;    -   R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, alkyl,        alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino,        alkylsulfonyl, alkylsulfonamido, sulfonamidoalkyl,        sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,        aminocarbonyl, aryl and heteroaryl;    -   R₁₅ is chosen from alkyl, aryl, alkylaryl, heteroaryl,        alkylheteroaryl, substituted alkyl, substituted aryl,        substituted alkylaryl, substituted heteroaryl, and substituted        alkylheteroaryl;    -   R₁₆ is chosen from alkoxy, amino, alkylamino, dialkylamino,        N-heterocyclyl and substituted N-heterocyclyl.

All of the compounds falling within the foregoing parent genus and itssubgenera are useful as kinesin inhibitors, but not all the compoundsare novel. In particular, certain ureas (i.e. compounds in which R₃ isR₁₅NH) are disclosed in U.S. Pat. No, 5,756,502 as agents which modifycholecystokinin action. The specific exceptions in the claims reflectapplicants' intent to avoid claiming subject matter that, whilefunctionally part of the inventive concept, is not patentable to themfor reasons having nothing to do with the scope of the invention.

Definitions

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns (e.g., substitutedalkyl includes optionally substituted cycloalkyl groups, which in turnare defined as including optionally substituted alkyl groups,potentially ad infinitum) that are sterically impractical and/orsynthetically non-feasible.

Alkyl is intended to include linear, branched, or cyclic hydrocarbonstructures and combinations thereof. Lower alkyl refers to alkyl groupsof from 1 to 5 carbon atoms. Examples of lower alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like.Preferred alkyl groups are those of C₂₀ or below. More preferred alkylgroups are those of C₁₃ or below. Cycloalkyl is a subset of alkyl andincludes cyclic hydrocarbon groups of from 3 to 13 carbon atoms.Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl,norbornyl, adamantyl and the like. In this application, alkyl refers toalkanyl, as well as the unsaturated alkenyl and alkynyl residues; it isintended to include cyclohexylmethyl, vinyl, allyl, isoprenyl and thelike. Alkylene refers to the same residues as alkyl, but having twopoints of attachment. Examples of alkylene include ethylene (—CH₂CH₂—),ethenylene (—CH₂═CH₂—), propylene (—CH₂CH₂CH₂—), dimethylpropylene(—CH₂C(CH₃)₂CH₂—) and cyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When analkyl residue having a specific number of carbons is named, allgeometric isomers having that number of carbons are intended to beencompassed; thus, for example, “butyl” is meant to include n-butyl,sec-butyl, isobutyl and t-butyl; “propyl” includes n-propyl andisopropyl (or “i-propyl”).

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of astraight, branched, cyclic configuration and combinations thereofattached to the parent structure through an oxygen. Examples includemethoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy andthe like. Lower-alkoxy refers to groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. Lower-acyl refers to groups containingone to four carbons.

Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromaticring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9-or 10-membered aromatic or heteroaromatic ring system containing 0-3heteroatoms selected from O, N, or S; or a tricyclic 13- or 14-memberedaromatic or heteroaromatic ring system containing 0-3 heteroatomsselected from O, N, or S. The aromatic 6- to 14-membered carbocyclicrings include, e.g., benzene, naphthalene, indane, tetralin, andfluorene and the 5- to 10-membered aromatic heterocyclic rings include,e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole,furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,pyrazine, tetrazole and pyrazole.

Alkylaryl refers to a residue in which an aryl moiety is attached to theparent structure via an alkylene residue. Examples are benzyl,phenethyl, phenylvinyl, phenylallyl and the like. Oxaalkyl andoxaalkylaryl refer to alkyl and alkylaryl residues in which one or moremethylenes have been replaced by oxygen. Examples of oxaalkyl andoxaalkylaryl residues are ethoxyethoxyethyl (3,6-dioxaoctyl),benzyloxymethyl and phenoxymethyl; in general, glycol ethers, such aspolyethyleneglycol, are intended to be encompassed by this group.Alkylheteroaryl refers to a residue in which a heteroaryl moiety isattached to the parent structure via an alkylene residue. Examplesinclude furanylmethyl, pyridinylmethyl, pyrimidinylethyl and the like.

Heterocycle means a cycloalkyl or aryl residue in which one to four ofthe carbons is replaced by a heteroatom such as oxygen, nitrogen orsulfur. Examples of heterocycles that fall within the scope of theinvention include imidazoline, pyrrolidine, pyrazole, pyrrole, indole,quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran,benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl,when occurring as a substituent), tetrazole, morpholine, thiazole,pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline,isoxazole, dioxane, tetrahydrofuran and the like. “N-heterocyclyl”refers to a nitrogen-containing heterocycle as a substituent residue.The term heterocyclyl encompasses heteroaryl, which is a subset ofheterocyclyl. Examples of N-heterocyclyl residues include 4-morpholinyl,4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl,piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examples of substitutedheterocyclyl include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl.

Substituted alkyl, aryl and heteroaryl refer to alkyl, aryl orheteroaryl wherein one or more H atoms are replaced with alkyl, halogen,hydroxy, alkoxy, alkylenedioxy (e.g., methylenedioxy), fluoroalkyl,carboxy (—COOH), carboalkoxy (i.e., acyloxy —O(O)CR), carboxyalkyl(i.e., esters —C(O)OR), carboxamido, sulfonamidoalkyl, sulfonamidoaryl,aminocarbonyl, benzyloxycarbonylamino (CBZ-amino), cyano, carbonyl,nitro, primary-, secondary- and tertiary-amino (e.g., alkylamino anddialkylamino) and aminoaklylene, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylsulfonamido, arylthio, arylsulfinyl, arylsulfonyl,amidino, aryl (e.g., phenyl and benzyl), heteroaryl, heterocyclyl,phenoxy, benzyloxy, or heteroaryloxy. For the purposes of the presentinvention, substituted alkyl also includes oxaalkyl residues, i.e. alkylresidues in which one or more carbons has been replaced by oxygen.Substituted alkylaryl and substituted oxaalkylaryl refer to residueswhere either or both of the alkylene and aryl moieties are substituted.Substituted alkylheteroaryl and substituted oxaalkylheteroaryl residueswhere either or both of the alkylene and heteroaryl moieties aresubstituted. It should additionally be noted that certain positions maycontain two or even three substitution groups, R, R′ and R″ (e.g.,diethylamino and trifluoromethyl).

Halogen refers to fluorine, chlorine, bromine or iodine. Fluorine,chlorine and bromine are preferred. Dihaloaryl, dihaloalkyl,trihaloaryl, etc. refer to aryl and alkyl substituted with a pluralityof halogens, but not necessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

Most of the compounds described herein contain one or more asymmetriccenters (e.g. the carbon to which R₂ and R₂′ are attached) and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)— or(S)—. The present invention is meant to include all such possibleisomers, including racemic mixtures, optically pure forms andintermediate mixtures. Optically active (R)- and (S)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

When desired, the R- and S-isomers may be resolved by methods known tothose skilled in the art, for example by formation of diastereoisomericsalts or complexes which may be separated, for example, bycrystallisation; via formation of diastereoisomeric derivatives whichmay be separated, for example, by crystallisation, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic oxidation orreduction, followed by separation of the modified and unmodifiedenantiomers; or gas-liquid or liquid chromatography in a chiralenvironment, for example on a chiral support, such as silica with abound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymnetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting on enantiomer to the other by asymmetrictransformation. An example of a synthesis from optically active startingmaterials is shown in FIG. 4.

In one embodiment, as will be appreciated by those in the art, the twoadjacent R₂ groups may be fused together to form a ring structure.Again, the fused ring structure may contain heteroatoms and may besubstituted with one or more substitution groups “R”. It shouldadditionally be noted that for cycloalkyl (i.e., saturated ringstructures), certain positions may contain two substitution groups, Rand R′.

Preferred Embodiments

Considering formulae 1a, 1b, 1c and 1d, but focusing on 1 a, in apreferred embodiment R₁ is selected from hydrogen, alkyl, aryl,substituted alkyl, substituted aryl, heteroaryl, substituted heteroaryl,alkylaryl and substituted alkylaryl.

In a more preferred embodiment R₁ is selected from hydrogen, loweralkyl,substituted lower alkyl, benzyl, substituted benzyl, phenyl,naphthyl and substituted phenyl.

In a most preferred embodiment R₁ is chosen from hydrogen, ethyl,propyl, methoxyethyl, naphthyl, phenyl, bromophenyl, chlorophenyl,methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl,methylchlorophenyl, ethylphenyl, phenethyl, benzyl, chlorobenzyl,methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl,tetrahydrofuranylmethyl and (ethoxycarbonyl)ethyl.

In a preferred embodiment R₂ is hydrogen, alkyl,cycloalkyl orsubstituted alkyl. As will be appreciated by those in the art, Formulae1a, 1b, 1c and 1d possess a potentially chiral center at the carbon towhich R₂ is attached. Thus, the R₂ position may comprise twosubstitution groups, R₂ and R₂′. The R₂ and R₂′ groups may be the sameor different; if different, the composition is chiral. When the R₂ andR₂′ are different, preferred embodiments utilize only a singlenon-hydrogen R₂. The invention contemplates the use of pure enantiomersand mixtures of enantiomers, including racemic mixtures, although theuse of the substantially optically pure eutomer will generally bepreferred, particularly the R enantiomer.

In a more preferred embodiment, R₂ is chosen from hydrogen, lower alkyland substituted lower alkyl, and R₂′ is hydrogen. In a most preferredembodiment R₂ is chosen from hydrogen, methyl, ethyl, propylparticularly i-propyl), butyl (particularly t-butyl), methylthioethyl,aminobutyl, (CBZ)aminobutyl, cyclohexylmethyl, benzyloxymethyl,methylsulfinylethyl, methylsulfinylmethyl, hydroxymethyl, benzyl andindolylmethyl. Especially preferred is the R enantiomer where R₂ isi-propyl.

In a preferred embodiment R₃ is selected from chosen from alkyl,substituted alkyl, alkylaryl, heteroaryl, aryl, substituted aryl,substituted oxaalkylaryl, —O—R₁₅ and —NH—R₁₅, and R₁₅ is chosen fromalkyl, aryl and substituted aryl.

In a more preferred embodiment, when R₃ is not —NHR₁₅, R₃ is chosen fromC₁-C₁₃ alkyl; substituted lower alkyl;aryl, including phenyl, biphenyland naphthyl; substituted aryl, including phenyl substituted with one ormore halo, lower alkyl, loweralkoxy, nitro, carboxy, methylenedioxy ortrifluoromethyl; benzyl; phenoxymethyl; halophenoxymethyl; phenylvinyl;heteroaryl; heteroaryl substituted with lower alkyl; andbenzyloxymethyl.

In a most preferred embodiment, when R₃ is not —NHR₁₅, R₃ is chosen fromethyl, propyl, chloropropyl, butoxy, heptyl, butyl, octyl, tridecanyl,(ethoxycarbonyl)ethyl, dimethylaminoethyl, dimethylaminomethyl, phenyl,naphthyl, halophenyl, dihalophenyl, cyanophenyl,halo(trifluoromethyl)phenyl, chlorophenoxymethyl, methoxyphenyl,carboxyphenyl, ethylphenyl, tolyl, biphenyl, methylenedioxyphenyl,methylsulfonylphenyl, methoxychlorophenyl, chloronaphthyl,methylhalophenyl, trifluoromethylphenyl, butylphenyl, pentylphenyl,methylnitrophenyl, phenoxymethyl, dimethoxyphenyl, phenylvinyl,nitrochlorophenyl, nitrophenyl, dinitrophenyl,bis(trifluoromethyl)phenyl, benzyloxymethyl, benzyl, furanyl,benzofuranyl, pyridinyl, indolyl, methylpyridinyl, quinolinyl,picolinyl, pyrazolyl, and imidazolyl.

In a more preferred embodiment, when R₃ is —NHR₁₅, R₁₅ is chosen fromlower alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, loweralkyl, loweralkoxy, or lower alkylthio.

In a most preferred embodiment, when R₃ is —NHR₁₅, R₁₅ is isopropyl,butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl,ethylphenyl, tolyl, trifluoromethylphenyl or methylthiophenyl.

In a preferred embodiment R₄ is chosen from alkyl, aryl, alkylaryl,alkylheteroaryl, substituted alkyl, substituted aryl, and -alkylene-R₁₆,and R₁₆ is chosen from alkoxy, amino, alkylamino, dialkylamino andN-heterocyclyl.

In a more preferred embodiment, R₄ is selected from lower alkyl,substituted lower alkyl, cyclohexyl; phenyl substituted with hydroxy,lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl;heteroarylpropyl and -alkylene-R₁₆, wherein R₁₆ is amino, loweralkylamino, di(lower alkyl)amino, lower alkoxy, or N-heterocyclyl.

In a most preferred embodiment, R₄ is chosen from methyl, ethyl, propyl,butyl, cyclohexyl, carboxyethyl, carboxymethyl, methoxyethyl,hydroxyethyl, hydroxypropyl, dimethylaminoethyl, dimethylaminopropyl,diethylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl,2,2-dimethyl-3-(dimethylamino)propyl,1-cyclohexyl-4-(diethylamino)butyl, aminoethyl, aminobutyl, aminopentyl,aminohexyl, aminoethoxyethyl, isopropylaminopropyl,diisopropylaminoethyl, 1-methyl-4-(diethylamino)butyl,(t-Boc)aminopropyl, hydroxyphenyl, benzyl, methoxyphenyl,methylmethoxyphenyl, dimethylphenyl, tolyl, ethylphenyl,(oxopyrrolidinyl)propyl, (methoxycarbonyl)ethyl, benzylpiperidinyl,pyridinylethyl, pyridinylmethyl, morpholinylethyl, morpholinylpropyl,piperidinyl, azetidinylmethyl, azetidinylpropyl, pyrrolidinylethyl,pyrrolidinylpropyl, piperidinylmethyl, piperidinylethyl,imidazolylpropyl, imidazolylethyl, (ethylpyrrolidinyl)methyl,(methylpyrrolidinyl)ethyl, (methylpiperidinyl)propyl,(methylpiperazinyl)propyl, furanylmethyl and indolylethyl.

In other preferred embodiments, R₅, R₆, R₇ and R₈ are chosen fromhydrogen, halo (particularly chloro and fluoro), lower alkyl(particularly methyl), substituted lower alkyl (particularlytrifluoromethyl), lower alkoxy (particularly methoxy), and cyano; morepreferably from hydrogen and halo. Further preferred for each of thespecific substituents: R₅ is hydrogen or halo; R₆ is hydrogen, methyl orhalo; R₇ is hydrogen, halo, alkyl (particularly methyl), alkoxy(particularly methoxy) or cyano; and R₈ is hydrogen or halo. Stillfurther preferred are the compounds where only one of R₅, R₆, R₇ and R₈is not hydrogen, especially R₇.

In a particularly preferred subgenus, R₁ is benzyl or halobenzyl; R₂ ischosen from ethyl and propyl; R₂′ is hydrogen; R₃ (or R₃ or R_(3″)) issubstituted phenyl; R₄ is —(CH₂)_(m)OH where m is two or three, or—(CH₂)_(p)R₁₆ where p is one to three and R₁₆ is amino, propylamino orazetidinyl; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo; and R₈ ishydrogen.

When considering primarily the sulfonamides of formula 1b, R₁ ispreferably chosen from hydrogen, lower alkyl, substituted lower alkyl,benzyl, substituted benzyl, phenyl and substituted phenyl; R₂ is chosenfrom hydrogen and lower alkyl and R₂′ is hydrogen; R_(3′) is chosen fromC₁-C₁₃ alkyl; phenyl; naphthyl; phenyl substituted with halo, loweralkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl;biphenylyl and heteroaryl; and R₄ is chosen from lower alkyl,cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or loweralkyl; benzyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl;heteroarylethyl; heteroarylpropyl and -alkylene-R₁₆, wherein R₁₆ isdi(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, orN-heterocyclyl, particularly pyrrolidino, piperidino or imidazolyl.

When considering primarily the sulfonamides of formula 1b, R₁ is mostpreferably chosen from lower allyl, benzyl, substituted benzyl andsubstituted phenyl; R₂ is hydrogen or lower alkyl; R₂′ is hydrogen; R₃is chosen from substituted phenyl and naphthyl; R₄ is -alkylene-R₁₆; R₇is hydrogen, fluoro, methyl or chloro; R₅, R₆ and R₈ are hydrogen; andR₁₆ is chosen from di(lower alkylamino), (lower alkyl)amino, amino,pyrrolidino, piperidino, imidazolyl and morpholino.

When considering primarily the amines of formulae 1c and 1d, R₁ ispreferably chosen from hydrogen, lower alkyl, substituted lower alkyl,benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R₂is chosen from hydrogen, lower alkyl and substituted lower alkyl and R₂′is hydrogen; R_(3″) is chosen from C₁-C₁₃ alkyl; substituted loweralkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl,lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl,benzyl and heterocyclyl; and R₄ is chosen from lower alkyl; cyclohexyl;phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl;substituted benzyl; heterocyclyl; heteroarylmethyl; heteroarylethyl;heteroarylpropyl and -alkylene-R₁₆, wherein R₁₆ is di(lower alkyl)amino,(lower alkyl)amino, amino, lower alkoxy, or N-heterocyclyl.

When considering primarily the amines of formulae 1c and 1d, R₁ is mostpreferably chosen from lower alkyl, benzyl, substituted benzyl andsubstituted phenyl; R₂ is hydrogen or lower alkyl; R₂′ is hydrogen;R_(3″) is chosen from substituted phenyl, heterocyclyl and naphthyl; R₄is chosen from subtituted benzyl, heterocyclyl and -alkylene-R₁₆; R₆ andR₇ are chosen from hydrogen and halo; R₅ and R₈ are hydrogen; and R₁₆ ischosen from di(lower alkylamino), (lower alkyl)amino, amino,pyrrolidinyl, piperidinyl, imidazolyl and morpholinyl. When R_(3″) ispresent (as in 1d) it is most preferably chosen from halophenyl,polyhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl,cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl,bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl,methoxycarbonylphenyl, piperidinyl and naphthyl.

In view of the foregoing, particularly when taken in consideration ofthe test data presented below, it will be appreciated that preferred forthe compounds, pharmaceutical formulations, methods of manufacture anduse of the present invention are the following combinations andpermutations of substituent groups (sub-grouped, respectively, inincreasing order of preference):

-   1. Any of formulae 1a, 1b, 1c or 1d (preferably formulae 1a or 1d)    where R₁is hydrogen, lower alkyl, substituted lower alkyl, alkylaryl    or substituted alkylaryl (preferably benzyl or substituted benzyl):    -   a. Especially where the stereogenic center to which R₂ and R₂′        are attached is of the R configuration, where R₂ is hydrogen.        -   i. Particularly where R₂ is lower alkyl (preferably ethyl,            i-proyl, c-propyl or t-butyl).            -   1. Most preferably where R₂ is i-propyl.    -   b. Especially those where R₄ is substituted alkyl, (preferably a        primary-, secondary- or tertiary-amino-substituted lower alkyl).        -   i. Most preferably where R₄ is primary-amino lower alkyl.    -   c. Especially those where R₅, R₆, R₇ and R₈ are chosen from        hydrogen, halo, lower alkyl (preferably methyl), substituted        lower alkyl, lower alkoxy (preferably methoxy), and cyano.        -   i. Preferably R₅, R₆, and R₈ are hydrogen.            -   1. More preferably R₇ is halo or cyano, especially                fluoro or chloro, and most preferably chloro.    -   d. In the case of formulae 1a and 1d, especially those where R₃        or R_(3″) is aryl (preferably phenyl), substituted aryl        (preferably lower alkyl- or lower alkoxy-substituted phenyl),        alkylaryl (preferably benzyl and phenylviny), alkylheteroaryl,        oxaalkylaryl (preferably phenoxy lower alkyl),        oxaalkylheteroaryl, substituted alkylaryl (preferably        substituted benzyl and substituted phenylviny), substituted        alkylheteroaryl, substituted oxaalkylaryl (preferably        substituted phenoxy lower alkyl), or substituted        oxaalkylheteroaryl.        -   i. Most preferably those where R₃ or R_(3″) is aryl,            substituted aryl, lower alkylaryl or substituted lower            alkylaryl.-   2. Any of formulae 1a, 1b, 1c or 1d, where the stereogenic center to    which R₂ and R₂′ are attached is of the R configuration,    particularly where R_(2′) is hydrogen:    -   a. Especially where R₂ is lower alkyl (preferably ethyl,        i-proyl, c-propyl or t-butyl).        -   i. Most preferably where R₂ is i-propyl.    -   b. Especially where R₄ is substituted alkyl (preferably a        primary-, secondary- or tertiary-amino-substituted lower alkyl).        -   i. Most preferably where R₄ is primary amino-lower alkyl.    -   c. Especially where R₅, R₆, and R₈ are hydrogen.        -   i. Most preferably where R₇ is hydrogen, halo (particularly            chloro or fluoro), lower alkyl (particularly methyl),            substituted lower alkyl, lower alkoxy (particularly            methoxy), or cyano            -   1. Especialy where R₇ is chloro.-   3. Any of formulae 1a, 1b, 1c or 1d where R₇ is hydrogen, halo    (preferably chloro or fluoro), lower alkyl (preferably methyl),    substituted lower alkyl, lower alkoxy (preferably methoxy), or    cyano.    -   a. Especially those where R₇ is halo or cyano.    -   b. Especially those where R₅, R₆, and R₈ are hydrogen.        -   i. Most preferably those where R₇ is chloro.-   4. Any of formulae 1b or 1c, where R₄ is substituted alkyl    (preferably a primary-, secondary- or tertiary-amino-substituted    lower alkyl, especially primary-amino lower alkyl).    -   a. Especially where the stereogenic center to which R₂ and R₂′        are attached is of the R configuration, particularly where        R_(2′) is hydrogen:        -   i. Especially where R₂ is lower alkyl (preferably ethyl,            i-proyl, c-propyl or t-butyl).            -   1. Most preferably where R₂ is i-propyl.    -   b. Especially where R₅, R₆, and R₈ are hydrogen.        -   i. Most preferably where R₇ is hydrogen, halo (particularly            chloro or fluoro), lower alkyl particularly methyl),            substituted lower alkyl, lower alkoxy (particularly            methoxy), or cyano            -   1. Especialy where R₇ is chloro.

Most preferred for the compounds, pharmaceutical formulations, methodsof manufacture and use of the present invention is formula 1aincorporating the following combinations and permutations of substituentgroups (sub-grouped, respectively, in increasing order of preference):

-   1. R₁ is alkylaryl or substituted alkylaryl (preferably benzyl or    substituted benzyl; most preferably benzyl).    -   a. Especially where the stereogenic center to which R₂ and R₂′        are attached is of the R configuration, where R₂′ is hydrogen.        -   i. Particularly those where R₂ is lower alkyl (preferably            ethyl, i-propyl, c-propyl or t-butyl).            -   1. Most preferably those where R₂ is i-propyl.    -   b. Especially those where R₃ is aryl (preferably phenyl),        substituted aryl (preferably lower alkyl-, lower alkoxy- and/or        halo-substituted phenyl), alkylaryl (preferably benzyl and        phenylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy        lower alkyl), oxaalkylheteroaryl, substituted alkylaryl        (preferably substituted benzyl and substituted phenylviny),        substituted alkylheteroaryl, substituted oxaalkylaryl        (preferably substituted phenoxy lower alkyl), or substituted        oxaalkylheteroaryl.        -   i. Particularly those where R₃ is aryl, substituted aryl,            lower alkylaryl, substituted lower alkylaryl,            oxa(lower)alkylaryl.            -   1. Most preferably those where R₃ is methyl- and/or                halo-substituted phenyl.    -   c. Especially those where R₄ is substituted alkyl (preferably a        primary-, secondary- or tertiary-amino-substituted lower alkyl).        -   i. Particularly those where R₄ is a            primary-amino-substituted lower alkyl.            -   1. Most preferably those where R₄ is 3-amino-n-propyl.    -   d. Especially those where R₅, R₆, R₇ and R₈ are chosen from        hydrogen, halo (preferably chloro and fluoro), lower alkyl        (preferably methyl), substituted lower alkyl, lower alkoxy        (preferably methoxy), and cyano.        -   i. Particularly those where R₅, 6, R₇ and R₈ are hydrogen,            halo, lower alkyl or cyano.            -   1. Most preferably those where R₅, R₆ and R₈ are                hydrogen.                -   a. Especially those where R₇ is halo or cyano (most                    preferably chloro).            -   2. Especially those where R₇ is halo or cyano (most                preferably chloro).-   2. Where the stereogenic center to which R₂ and R₂′ are attached is    of the R configuration (preferably where R₂′ is hydrogen).    -   a. Especially those where R₂ is lower alkyl (preferably ethyl,        i-propyl, c-propyl or t-butyl).        -   i. Most preferably those where R₂ is i-propyl.    -   b. Especially those where R₃ is aryl (preferably phenyl),        substituted aryl (preferably lower alkyl-, lower alkoxy-, and/or        halo-substituted phenyl), alkylaryl (preferably benzyl and        phenylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy        lower alkyl), oxaalkylheteroaryl, substituted alkylaryl        (preferably substituted benzyl and substituted phenylviny),        substituted alkylheteroaryl, substituted oxaalkylaryl        (preferably substituted phenoxy lower alkyl), or substituted        oxaalkylheteroaryl.        -   i. Particularly those where R₃ is aryl, substituted aryl,            lower alkylaryl, substituted lower alkylaryl,            oxa(lower)alkylaryl.            -   1. Most preferably those where R₃ is methyl- and/or                halo-substituted phenyl.    -   c. Especially those where R₄ is substituted alkyl (preferably a        primary-, secondary- or tertiary-amino-substituted lower alkyl).        -   i. Particularly those where R₄ is a            primary-amino-substituted lower alkyl.            -   1. Most preferably those where R₄ is 3-amino-n-propyl.    -   d. Especially those where R₅, R₆, R₇ and R₈ are chosen from        hydrogen, halo (preferably chloro and fluoro), lower alkyl        (preferably methyl), substituted lower alkyl, lower alkoxy        (preferably methoxy), and cyano.        -   i. Particularly those where R₅, R₆, R₇ and R₈ are hydrogen,            halo or lower alkyl.            -   1. Most preferably those where R₅, R₆ and R₈ are                hydrogen.                -   a. Especially those where R₇ is halo or cyano (most                    preferably chloro).            -   2. Especially those where R₇ is halo or cyano (most                preferably chloro).-   3. R₃ is aryl (preferably phenyl), substituted aryl (preferably    lower alkyl-, lower alkoxy-, and/or halo-substituted phenyl),    alkylaryl (preferably benzyl and phenylviny), alkylheteroaryl,    oxaalkylaryl (preferably phenoxy lower alkyl), oxaalkylheteroaryl,    substituted alkylaryl (preferably substituted benzyl and substituted    phenylviny), substituted alkylheteroaryl, substituted oxaalkylaryl    (preferably substituted phenoxy lower alkyl), or substituted    oxaalkylheteroaryl.    -   a. Especially those where R₃ is aryl, substituted aryl, lower        alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaryl.        -   i. Most preferably those where R₃ is methyl- and/or            halo-substituted phenyl.    -   b. Especially those where R₄ is substituted alkyl (preferably a        primary-, secondary- or tertiary-amino-substituted lower alkyl).        -   i. Particularly those where R₄ is a            primary-amino-substituted lower alkyl.            -   1. Most preferably those where R₄ is 3-amino-n-propyl.    -   c. Especially those where R₅, R₆, R₇ and R₈ are chosen from        hydrogen, halo (preferably chloro and fluoro), lower alkyl        (preferably methyl), substituted lower alkyl, lower alkoxy        (preferably methoxy), and cyano.        -   i. Particularly those where R₅, R₆, R₇ and R₈ are hydrogen,            halo, lower alkyl or cyano.            -   1. Most preferably those where R₅, R₆ and R₈ are                hydrogen.                -   a. Especially those where R₇ is halo or cyano (most                    preferably chloro).            -   2. Especially those where R₇ is halo or cyano (most                preferably chloro).-   4. R₄ is substituted alkyl (preferably a primary-, secondary- or    tertiary-amino-substituted lower alkyl).    -   a. Particularly those where R₄ is a primary-amino-substituted        lower alkyl.        -   i. Most preferably those where R₄ is 3-amino-n-propyl.    -   b. Especially those where R₅, R₆, R₇ and R₈ are chosen from        hydrogen, halo (preferably chloro and fluoro), lower alkyl        (preferably methyl), substituted lower alkyl, lower alkoxy        (preferably methoxy), and cyano.        -   i. Particularly those where R₅, R₆, R₇ and R₈ are hydrogen,            halo, lower alkyl or cyano.            -   1. Most preferably those where R₅, R₆ and R₈ are                hydrogen.                -   a. Especially those where R₇ is halo or cyano (most                    preferably chloro).            -   2. Especially those where R₇ is halo or cyano (most                preferably chloro).-   5. R₅, R₆, R₇ and R₈ are chosen from hydrogen, halo (preferably    chloro and fluoro), lower alkyl (preferably methyl), substituted    lower alkyl, lower alkoxy (preferably methoxy), and cyano.    -   a. Especially those where R₅, R₆, R₇ and R₈ are hydrogen, halo,        lower alkyl or cyano.        -   i. Most preferably those where R₅, R₆ and R₈ are hydrogen.            -   1. Especially those where R₇ is halo or cyano (most                preferably chloro).        -   ii. Especially those where R₇ is halo or cyano (most            preferably chloro).

Especially preferred for the compounds, pharmaceutical formulations,methods of manufacture and use of the present invention is formula 1awhere R₁ is alkylaryl or substituted alkylaryl (particularly benzyl orsubstituted benzyl), R₂ is lower alkyl (particularly i-propyl), R₂′ ishydrogen, R₃ is substituted aryl (particularly methyl- and/orhalo-substituted phenyl), R₄ is substituted alkyl (particularly3-amino-n-propyl), R₅, R₆ and R₈ are hydrogen, and R₇ is halo or cyano(particularly chloro), where the stereogenic center to which R₂ and R₂′are attached is of the R configuration.

Synthesis, Testing and Use

The compositions of the invention are synthesized as outlined below,utilizing techniques well known in the art. For example, as described inAger et al., J. of Med. Chem., 20:379-386 (1977), hereby incorporated byreference, quinazolinones can be obtained by acid-catalyzed condensationof N-acylanthranilic acids with aromatic primary amines. Other processesfor preparing quinazolinones are described in U.S. Pat. Nos. 5,783,577,5,922,866 and 5,187,167, all of which are incorporated by reference.

The compositions of the invention may be made as shown in FIGS. 1, 2, 4and 5. Compounds of formulae 1d are made in analogous fashion to FIG. 1,except that the acyl halide in the final step is replaced by an alkylhalide.

Once made, the compositions of the invention find use in a variety ofapplications. As will be appreciated by those in the art, mitosis may bealtered in a variety of ways; that is, one can affect mitosis either byincreasing or decreasing the activity of a component in the mitoticpathway. Stated differently, mitosis may be affected (e.g., disrupted)by disturbing equilibrium, either by inhibiting or activating certaincomponents. Similar approaches may be used to alter meiosis.

In a preferred embodiment, the compositions of the invention are used tomodulate mitotic spindle formation, thus causing prolonged cell cyclearrest in mitosis. By “modulate” herein is meant altering mitoticspindle formation, including increasing and decreasing spindleformation. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compositions of the invention are useful to bind to and/or modulatethe activity of a mitotic kinesin, KSP. In a preferred embodiment, theKSP is human KSP, although KSP kinesins from other organisms may also beused. In this context, modulate means either increasing or decreasingspindle pole separation, causing malformation, i.e., splaying, ofmitotic spindle poles, or otherwise causing morphological perturbationof the mitotic spindle. Also included within the definition of KSP forthese purposes are variants and/or fragments of KSP. See U.S. patentapplication “Methods of Screening for Modulators of Cell Proliferationand Methods of Diagnosing Cell Proliferation States”, filed Oct. 27,1999 (U.S. Ser. No. 09/428,156), hereby incorporated by reference in itsentirety. In addition, other mitotic kinesins may be used in the presentinvention. However, the compositions of the invention have been shown tohave specificity for KSP.

For assay of activity, generally either KSP or a compound according tothe invention is non-diffusably bound to an insoluble support havingisolated sample receiving areas (e.g., a microtiter plate, an array,etc.). The insoluble support may be made of any composition to which thecompositions can be bound, is readily separated from soluble material,and is otherwise compatible with the overall method of screening. Thesurface of such supports may be solid or porous and of any convenientshape. Examples of suitable insoluble supports include microtiterplates, arrays, membranes and beads. These are typically made of glass,plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,Teflon™, etc. Microtiter plates and arrays are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples. The particular manner ofbinding of the composition is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies (which do not sterically blockeither the ligand binding site or activation sequence when the proteinis bound to the support), direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or agent, excess unboundmaterial is removed by washing. The sample receiving areas may then beblocked through incubation with bovine serum albumin (BSA), casein orother innocuous protein or other moiety.

The antimitotic agents of the invention may be used on their own tomodulate the activity of a mitotic kinesin, particularly KSP. In thisembodiment, the mitotic agents of the invention are combined with KSPand the activity of KSP is assayed. Kinesin activity is known in the artand includes one or more kinesin activities. Kinesin activities includethe ability to affect ATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes; such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

Methods of performing motility assays are well known to those of skillin the art. [See e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476,Turner et al., 1996, AnaL Biochem. 242 (1):20-5; Gittes et al., 1996,Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J. Exp. BioL 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann etal., 1995, Biophys. J. 68: 72S.]

Methods known in the art for determining ATPase hydrolysis activity alsocan be used. Preferably, solution based assays are utilized. U.S.application Ser. No. 09/314,464, filed May 18, 1999, hereby incorporatedby reference in its entirety, describes such assays. Alternatively,conventional methods are used. For example, P_(i) release from kinesincan be quantified. In one preferred embodiment, the ATPase hydrolysisactivity assay utilizes 0.3 M PCA (perchloric acid) and malachite greenreagent (8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate,and 0.8 mM Triton X-1 00). To perform the assay, 10 μL of reaction isquenched in 90 μL of cold 0.3 M PCA. Phosphate standards are used sodata can be converted to mM inorganic phosphate released. When allreactions and standards have been quenched in PCA, 100 μL of malachitegreen reagent is added to the relevant wells in e.g., a microtiterplate. The mixture is developed for 10-15 minutes and the plate is readat an absorbance of 650 nm. If phosphate standards were used, absorbancereadings can be converted to mM P_(i) and plotted over time.Additionally, ATPase assays known in the art include the luciferaseassay.

ATPase activity of kinesin motor domains also can be used to monitor theeffects of modulating agents. In one embodiment ATPase assays of kinesinare performed in the absence of microtubules. In another embodiment, theATPase assays are performed in the presence of microtubules. Differenttypes of modulating agents can be detected in the above assays. In apreferred embodiment, the effect of a modulating agent is independent ofthe concentration of microtubules and ATP. In another embodiment, theeffect of the agents on kinesin ATPase can be decreased by increasingthe concentrations of ATP, microtubules or both. In yet anotherembodiment, the effect of the modulating agent is increased byincreasing concentrations of ATP, microtubules or both.

Agents that modulate the biochemical activity of KSP in vitro may thenbe screened in vivo. Methods for such agents in vivo include assays ofcell cycle distribution, cell viability, or the presence, morphology,activity, distribution, or amount of mitotic spindles. Methods formonitoring cell cycle distribution of a cell population, for example, byflow cytometry, are well known to those skilled in the art, as aremethods for determining cell viability. See for example, U.S. patentapplication “Methods of Screening for Modulators of Cell Proliferationand Methods of Diagnosing Cell Proliferation States,” filed Oct. 22,1999, Ser. No. 09/428,156, hereby incorporated by reference in itsentirety.

In addition to the assays described above, microscopic methods formonitoring spindle formation and malformation are well known to those ofskill in the art (see, e.g., Whitehead and Rattner (1998), J. Cell Sci.111:2551-61; Galgio et al, (1996) J. Cell biol., 135:399-414).

The compositions of the invention inhibit the KSP kinesin. One measureof inhibition is IC₅₀, defined as the concentration of the compositionat which the activity of KSP is decreased by fifty percent. Preferredcompositions have IC₅₀'s of less than about 1 mM, with preferredembodiments having IC₅₀'s of less than about 100 μM, with more preferredembodiments having IC₅₀'s of less than about 10 μM, with particularlypreferred embodiments having IC₅₀'s of less than about 1 μM, andespecially preferred embodiments having IC₅₀'s of less than about 100nM, and with the most preferred embodiments having IC₅₀'s of less thanabout 10 nM. Measurement of IC₅₀ is done using an ATPase assay.

Another measure of inhibition is K_(i). For compounds with IC₅₀'s lessthan 1 μM, the K_(i) or K_(d) is defined as the dissociation rateconstant for the interaction of the quinazolinone with KSP. Preferredcompounds have K_(i)'s of less than about 100 μM, with preferredembodiments having K_(i)'s of less than about 10 μM, and particularlypreferred embodiments having K_(i)'s of less than about 1 μM andespecially preferred embodiments having K_(i)'s of less than about 100nM, and with the most preferred embodiments having K_(i)'s of less thanabout 10 nM. The K_(i) for a compound is determined from the IC₅₀ basedon three assumptions. First, only one compound molecule binds to theenzyme and there is no cooperativity. Second, the concentrations ofactive enzyme and the compound tested are known (i.e., there are nosignificant amounts of impurities or inactive forms in thepreparations). Third, the enzymatic rate of the enzyme-inhibitor complexis zero. The rate (i.e., compound concentration) data are fitted to theequation:

$V = {V_{\max}{E_{0}\left\lbrack {I - \frac{\left( {E_{0} + I_{0} + {Kd}} \right) - \sqrt{\left( {E_{0} + I_{0} + {Kd}} \right)^{2} - {4\; E_{0}I_{0}}}}{2E_{0}}} \right\rbrack}}$

Where V is the observed rate, V_(max) is the rate of the free enzyme, I₀is the inhibitor concentration, E₀ is the enzyme concentration, andK_(d) is the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is GI₅₀, defined as the concentration ofthe compound that results in a decrease in the rate of cell growth byfifty percent. Preferred compounds have GI₅₀'s of less than about 1 mM.The level of preferability of embodiments is a function of their GI₅₀:those having GI₅₀'s of less than about 20 μM are more preferred; thosehaving GI₅₀'s of 10 μM more so; those having GI₅₀ of less than about 1μM more so; those having GI₅₀'s of 100 nM more so; those having GI₅₀ ofless than about 10 nM even more so. Measurement of GI₅₀ is done using acell proliferation assay.

The compositions of the invention are used to treat cellularproliferation diseases. Disease states which can be treated by themethods and compositions provided herein include, but are not limitedto, cancer (further discussed below), autoimmune disease, arthritis,graft rejection, inflammatory bowel disease, proliferation induced aftermedical procedures, including, but not limited to, surgery, angioplasty,and the like. It is appreciated that in some cases the cells may not bein a hyper or hypo proliferation state (abnormal state) and stillrequire treatment. For example, during wound healing, the cells may beproliferating “normally”, but proliferation enhancement may be desired.Similarly, as discussed above, in the agriculture arena, cells may be ina “normal” state, but proliferation modulation may be desired to enhancea crop by directly enhancing growth of a crop, or by inhibiting thegrowth of a plant or organism which adversely affects the crop. Thus, inone embodiment, the invention herein includes application to cells orindividuals afflicted or impending affliction with any one of thesedisorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); G necological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma],fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the mitotic agents of the invention toa cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Mitotic agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agents may be administered alone orin combination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts. “Pharmaceuticallyacceptable acid addition salt” refers to those salts that retain thebiological effectiveness of the free bases and that are not biologicallyor otherwise undesirable, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the mitotic agents of the present invention can bedone in a variety of ways as discussed above, including, but not limitedto, orally, subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the anti-mitotic agents may be directly appliedas a solution or spray.

To employ the compounds of the invention in a method of screening forcompounds that bind to KSP kinesin, the KSP is bound to a support, and acompound of the invention (which is a mitotic agent) is added to theassay. Alternatively, the compound of the invention is bound to thesupport and KSP is added. Classes of compounds among which novel bindingagents may be sought include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of particular interest are screening assays for candidate agentsthat have a low toxicity for human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, functional assays (phosphorylation assays, etc.) andthe like.

The determination of the binding of the mitotic agent to KSP may be donein a number of ways. In a preferred embodiment, the mitotic agent (thecompound of the invention) is labeled, for example, with a fluorescentor radioactive moiety and binding determined directly. For example, thismay be done by attaching all or a portion of KSP to a solid support,adding a labeled mitotic agent (for example a compound of the inventionin which at least one atom has been replaced by a detectable isotope),washing off excess reagent, and determining whether the amount of thelabel is that present on the solid support. Various blocking and washingsteps may be utilized as is lcnown in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the mitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate bioactive agent” or “drugcandidate” or grammatical equivalents as used herein describe anymolecule, e.g., protein, oligopeptide, small organic molecule,polysaccharide, polynucleotide, etc., to be tested for bioactivity. Theymay be capable of directly or indirectly altering the cellularproliferation phenotype or the expression of a cellular proliferationsequence, including both nucleic acid sequences and protein sequences.In other cases, alteration of cellular proliferation protein bindingand/or activity is screened. Screens of this sort may be performedeither in the presence or absence of microtubules. In the case whereprotein binding or activity is screened, preferred embodiments excludemolecules already known to bind to that particular protein, for example,polymer structures such as microtubules, and energy sources such as ATP.Preferred embodiments of assays herein include candidate agents which donot bind the cellular proliferation protein in its endogenous nativestate termed herein as “exogenous” agents. In another preferredembodiment, exogenous agents further exclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrinidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

Competitive screening assays may be done by combining KSP and a drugcandidate in a first sample. A second sample comprises a mitotic agent,KSP and a drug candidate. This may be performed in either the presenceor absence of microtubules. The binding of the drug candidate isdetermined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to KSP and potentially modulating its activity. That is, if thebinding of the drug candidate is different in the second sample relativeto the first sample, the drug candidate is capable of binding to KSP.

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to KSP for a timesufficient to allow binding, if present. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C.

Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to KSP and thus is capable of binding to, andpotentially modulating, the activity of KSP. In this embodiment, eithercomponent can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate agent islabeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

It may be of value to identify the binding site of KSP. This can be donein a variety of ways. In one embodiment, once KSP has been identified asbinding to the mitotic agent, KSP is fragmented or modified and theassays repeated to identify the necessary components for binding.

Modulation is tested by screening for candidate agents capable ofmodulating the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Abbreviations and Definitions

The following abbreviations and terms have the indicated meaningsthroughout:

-   Ac=acetyl-   BNB=4-bromomethyl-3-nitrobenzoic acid-   Boc=t-butyloxy carbonyl-   Bu=butyl-   c-=cyclo-   CBZ=carbobenzoxy=benzyloxycarbonyl-   DBU=diazabicyclo[5.4.0]undec-7-ene-   DCM=dichloromethane=methylene chloride CH₂C₁₂-   DCE dichloroethylene-   DEAD=diethyl azodicarboxylate-   DIC=diisopropylcarbodiimide-   DIEA=N,N-diisopropylethyl amine-   DMAP=4-N,N-dimethylaminopyridine-   DMF=N,N-dimethylformamide-   DMSO dimethyl sulfoxide-   DVB=1,4-divinylbenzene-   EEDQ=2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline-   Et ethyl-   Fmoc=9-fluorenylmethoxycarbonyl-   GC=gas chromatography-   HATU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HMDS=hexamethyldisilazane-   HOAc=acetic acid-   HOBt=hydroxybenzotriazole-   Me=methyl-   mesyl=methanesulfonyl-   MTBE=methyl t-butyl ether-   NMO=N-methylmorpholine oxide-   PEG=polyethylene glycol-   Ph=phenyl-   PhOH=phenol-   Pfp=pentafluorophenol-   PPTS=pyridinium p-toluenesulfonate-   Py=pyridine-   PyBroP=bromo-tris-pyrrolidino-phosphonium hexafluorophosphate-   rt=room temperature-   sat=d=saturated-   s-=secondary-   t-=tertiary-   TBDMS=t-butyldimethylsilyl-   TES=triethylsilane-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran-   TMOF=trimethyl orthoformate-   TMS=trimethylsilyl-   tosyl=p-toluenesulfonyl-   Trt=triphenylmethyl

Example 1 Synthesis of Compounds

The general synthesis is shown in FIGS. 1 and 2.

Step 1: N-butyryl anthranilic acid.

To a three-necked, 500 mL round-bottom flask equipped with athermometer, dropping funnel, and an efficient magnetic stir bar, wasadded anthranilic acid (1) (0.5 mole, 68.5 g) and dimethyl formamide(250 mL). To this solution was added butyryl chloride (0.55 mole, 57.1mL) dropwise at such a rate that the temperature of the mixture did notrise above 40° C. The suspension was stirred vigorously at roomtemperature for at least an additional 3 h. The mixture was poured intowater (2000 mL) and stirred for another 1 h. The precipitated productwas collected by filtration, washed with cold water, and dried underreduced pressure over P₂0₅, yielding compound 2 (67.3 g, 65%).

Step 2: 2-Propyl-3,1-[4H]benzoxazin-4-one.

Compound 2 (51.8 g, 0.25 mole) was dissolved in acetic anhydride (180mL) in a 500 mL round-bottom flask equipped with a magnetic stir bar, aClaisen-distillation head (with vacuum inlet) and a thermometer. Theflask was placed in an oil bath and slowly heated to 170-180° C. withvigorous stirring. The acetic acid produced was slowly distilled offunder atmospheric pressure. Monitoring the head temperature of thedistillation unit was used to follow the progress of the transformation.The reaction mixture was then cooled to 60° C. and the excess of aceticanhydride removed by distillation under reduced pressure (ca. 20 mm Hg).The residue was afterward cooled and the product crystallized. Theproduct was triturated with n-hexane (75 mL) and isolated by filtrationto yield 2-propyl-3,1-[4H]benzoxazin-4-one (3) (29.3, 62%). The aboveprocedure gave compound 3 sufficiently pure to use directly in the nextstep.

Step 3: 2-Propyl-3-benzylquinazolin-4-one.

Compound 3 (28.4 g, 0.15 mole) and benzylamine (17.5 mL, 0.16 mole) wererefluxed in chloroform (50 ml) in a one-neck 250 mL round-bottom flaskfor 6 h. After complete consumption of compound 3, the chloroform wasevaporated under reduced pressure. Ethylene glycol (100 mL) and NaOHpellets (0.60 g) were added to the residue and the flask equipped with aClaisen-distillation head and a magnetic stir bar. The flask wasimmersed in an oil bath and reheated to 130-140° C. bath temperaturewith vigorous stirring and maintained there for 5 h while the waterproduced was removed by distillation. After completion of the reaction,the clear solution was allowed to cool to room temperature and keptovernight to precipitate the product. The pH of the suspension wasadjusted to 7-8 by adding 3% aq. HCl, the crystals were filtered off andwashed with cold water, and then recrystallized from isopropanol (oralternatively from acetone) to provide the compound,2-propyl-3-benzylquinazolin-4-one (compound 4) (28.0 g, 67%).

Step 4: 2-(1′-bromopropyl)-3-benzylguinazolin-4-one.

To a three-neck 250 mL round-bottom flask equipped with a thermometer,dropping funnel, and efficient magnetic stir bar was added compound 4(27.8 g, 0.10 mole), anhydrous sodium acetate (10.0 g) and glacialacetic acid (130 mL). Bromine (16.0 g, 0.10 mole) dissolved in aceticacid (10 mL) was added dropwise to the above solution at 40° C. for 1-2h. After addition was complete, the mixture was poured into water (1500mL) and stirred for 1-2 h at room temperature. The precipitated product,2-(1′-bromopropyl)-3-benzylquinazolin-4-one (5) was isolated byfiltration, washed with warm water to remove traces of acetic acid, andrinsed with a small amount of isopropanol. Drying yielded compound 5(33.0 g, 92%).

Step 5:2-[1′-(N,N-dimethylethylenediamino)propyl]-3-benzylquinazolin-4-one.

Compound 5 (10.7 g, 0.03 mole) and N,N-dimethylethylenediamine (6.6 mL,0.06 mole) were dissolved in abs. ethanol (60 mL) and heated at refluxfor 6 h. After completion of the reaction, the solvent was evaporatedunder reduced pressure. The residue was dissolved in dichloromethane(150 mL) and washed with 3% aq. NaOH solution (ca. 10-20 mL). Theorganic layer was dried over MgSO₄ and evaporated to dryness underreduced pressure. The remaining oily product was purified by flashchromatography on a short silica gel pad using an eluent ofCHCl₃-MeOH-aq.NH₃, 90:10:0.1, to give the desired compound (5),2-[1′-(N,N-dimethylethylenediamino)propyl]-3-benzylquinazolin-4-one (6)(6.0 g, 55%).

Step 6:2-[1′-(N-4-fluorobenzoyl)-(N,N-dimethylethylenediamino)propyl]-3-benzylquinazolin-4-one.

A stock solution of compound 5 (1.822 g, 5.0 mmol) was prepared in HPLCgrade CHCl₃ (0.5 mL). A stock solution of p-flurobenzoyl chloride (160.2mg, 1 mmol) in HPLC grade 1,2-dichloroethane (2.0 mL) was prepared in a2.0 mL volumetric flask. A third solution of triethylamine (2.0 mL of0.5 M) was prepared in HPLC grade 1,2-dichlorethane. A 100 μL aliquot ofeach solution was pipetted into a glass reaction vessel using a BeckmanBiomet 2000 automated liquid dispenser. The reaction mixture was shakenusing a mechanical shaker, sonicated in an ultrasonic water bath, andthen incubated overnight at room temperature. The mixture was diluted inCHCl₃ (300 μL) and washed with 5% aqueous NaHCO₃ and water. The solventwas removed in vacuo to provide compound 6 (65%). The purity of thecompound was analyzed by TLC eluted with CH₂C₁₂-ethanol-concentratedaqueous NH₃, 100:10:1.

Examples 2 and 3 Synthesis of compounds of General Formula 1d

All anhydrous solvents were purchased from Aldrich chemical company inSureSea® containers. Most reagents were purchase from Aldrich ChemicalCompany. Abbreviations: DCM, dichloromethane; DIEA,N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; TES,triethylsilane; TFA, trifluoroacetic acid. Array synthesis was conductedin 15×75 mm glass round bottom screw-cap vials contained in a 4×6 arrayaluminum synthesis block, sealed with a Teflon-lined rubber membrane.Reagents were added and aqueous extractions performed with single ormultichannel pipettors. Filtrations were performed usingWhatman/Polyfiltronics 24 well, 10 mL filtration blocks. Evaporation ofvolatile materials from the array was performed with a LabconcoVortex-Evaporator or by sweeping with a 4×6 nitrogen manifold.

Example 2 Solid Phase Synthesis of a Single Compound

STEP 1: 1,3-Diaminopropane trityl resin (Novabiochem, 1.2 mmol/g) (0.20g, 0.24 mmol) was weighed into a screw-cap vial and 3 mL of a 1:1mixture of DMF and chloroform was added. DIEA (0.130 mL, 0.72 mmol) and2-(1′-bromopropyl)-3-benzylquinazolin-4-one (from Example 1) (0.188 g,0.48 mmol) were added. The vial was sealed, heated to 70° C. and shakenovernight. The resin was filtered and washed (3× DCM, 2× MeOH, 1× DCM,2× ether) and dried under vacuum. A 27 mg aliquot of resin was treatedwith 5:5:90 TFA:TES:DCM for 15 min and the mixture was filtered andevaporated, resulting in 8 mg (64% yield) of the quinazolinone-diamineintermediate. LCMS analysis showed >80% purity.

STEP 2: The resin from Step 1 was swelled in 3 mL of DCM. DIEA (0.130mL, 0.72 mmol) and 4-bromobenzyl bromide (0.12 g, 0.48 mmol) were added.The vial was sealed and shaken overnight. LCMS analysis of a cleavedaliquot revealed an approximate 1:1 mixture of starting material andproduct. Another 0.130 mL of DIEA and 0.12 g of 4-bromobenzyl bromidewere added and the mixture was shaken at 70° C. for 8 h. The resin wasfiltered, washed (as above), and dried under vacuum.

STEP 3: The resin from Step 2 was twice shaken for 30 min with 5:5:90TFA:TES:DCM and filtered. The filtrates were combined and evaporated,yielding 140 mg of an orange oil. This material was purified by reversephase preparative HPLC (acetonitrile-water gradient) to provide 27 mg(17% for 3 steps) of the mono-TFA salt.

Example 3 Combinatorial Synthesis of Multiple Compounds

STEP 1: 1,2-Diaminoethane trityl resin (Novabiochem, 0.95 mmol/g) (200g, 1.9 mmol) and 1,3-Diaminopropane trityl resin (Novabiochem, 1.14mmol/g) (2.0 g, 2.28 mmol) were each placed in different 10 mLpolypropylene fritted tubes (Bio-Rad). To each were added 4 mL of DMF, 4mL of chloroform, 3 eq. of DEA (1.0 mL and 1.2 mL, respectively) and 2eq. of 2-(1′-bromopropyl)-3-benzylquinazolin-4-one (from Example 1) (1.5g and 1.8 g, respectively). The mixtures were shaken at 70° C.overnight. Each mixture was washed (3× DCM, 2× MeOH, 1× DCM, 2× ether)and dried under vacuum. Analysis of a cleaved aliquot revealed thepresence of the appropriate quinazolinone-diamine for each in >90%purity.

STEP 2: The quinazolinone ethyl-diamine resin (105 mg, 0.10 mmol) wasplaced into each of the vials in the first 2 rows of the array, and thequinazolinone propyl-diamine resin (88 mg, 0.10 mmol) was placed intoeach vial of the last 2 rows of the array. To each vial was added DIEA(0.131 mL, 0.75 mmol). Into each vial of the first 2 rows of the arraywas added a different amine, and the additions were repeated for thelast two rows of the array. The reaction block was shaken at 70° C.overnight. Liquid was removed from each vial by multichannel pipetteusing fine-pointed gel-well tips, and the resins were washed (2× DCM, 1×MeOH, 1× DCM) and dried under vacuum.

STEP 3: To each vial of the array was added 2 mL of a 10:5:85TFA:TES:DCM solution. The reaction block was shaken for 45 min and themixtures were transferred to a filter block, filtered, and washed twicewith 0.75 mL DCM. The solutions were evaporated to yield yellow-to-redoils. These thick oils were triturated twice with ether, dissolved inDCM and treated with 4 M HCl in dioxane to provide the HCl salts(unknown number of salts per compound) as tan-to-white powdery oramorphous solids. Analysis by LCMS showed all to be >75% pure.

Examples 4-6

Six racemic quinazolinones were separated into their enantiomers bychiral chromatography. The chiral chromatography of three of thesecompounds is described below:

Example 4

Column—Chiralpak AD, 250×4.6 mm (Diacel Inc.). Sample—0.5 mg/mL in EtOH.

Conditions—15 min at 60% EtOH in Hexane, enantiomer 1 elutes at 4.5 min,enantiomer 2 elutes at 4.9 min.

Example 5

Column—Chiralcel OJ, 250×4.6 mm (Diacel Inc.). Sample—0.5 mg/mL in EtOH.

Conditions—15 min at 10% EtOH in Hexane, (R)-enantiomer elutes at 8.4min, (S)-enantiomer elutes at 9.6 min.

Example 6

Column—Chiralpak AD, 250×4.6 mm (Diacel Inc.). Sample—0.5 mg/mL in ETOH.

Conditions—15 min at 70% EtOH in Hexane, enantiomer 1 elutes at 6.5 min,enantiomer 2 elutes at 8.8 min.

The table below depicts the IC₅₀ activity of the racemate and theenantiomers of three other compounds separated as above. In all threecases, one enantiomer was significantly more potent than the other. Byindependent chiral synthesis, it appears that the more active enantiomeris the R enantiomer.

IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) Racemate Enantiomer 1 Enantiomer 2

0.06 0.28 0.03

12.7 >>40 6.6

2.6 >>40 1.3

Examples 7 and 8

The following two compounds were synthesized as single enantiomers bythe route shown in FIG. 4. The data indicate that the more activeenantiomer is the R enantiomer.

K_(i) (μM) K_(i) (μM) S enantiomer R enantiomer

2 <0.01

>0.5 <0.05

Example 9 Chiral Resolution by Recrystallization with Tartaric Acid

Intermediate A, prepared in Example 1, can be converted to anintermediate B, which, upon resolution, provides an alternative to thefirst five steps shown in FIG. 4. The process is shown in the schemebelow:

The R enantiomer of B can be crystallized selectively by heating amixture of B with 1.1 equivalents of D-tartaric acid in a mixture ofisopropanol and methanol and then letting the mixture return to roomtemperature.

Example 9: X═Cl, R═H

Racemic intermediate B (1.5 g), dissolved in 100 mL of boilingisopropanol, was mixed with 0.8 g of D-tartaric acid in 100 mL ofboiling methanol. The mixture was allowed to slowly reach roomtemperature. After standing overnight, the solid was removed byfiltration and rinsed with ethyl acetate and hexanes, and allowed to airdry. The dried solid (0.8 g) was then dissolved in a boiling mixture of50 mL of isopropanol and 50 mL of methanol and allowed to slowly cool toroom temperature. After standing overnight, the resulting solid wasremoved by filtration and rinsed with ethyl acetate and hexanes, andallowed to air dry. The dried solid was then stirred with saturatedsodium bicarbonate for 30 min and extracted with ethyl acetate. Theorganics were dried (MgSO₄), filtered and evaporated to dryness. Theresulting clear oil weighed 345 mg. Chiral purity of >95% was determinedby conversion of a portion to the S-Mosher amide and examination of theproduct by ¹HNMR. The enantiomerically pure compounds below wereprepared, according to the remaining steps in FIG. 4, from materialresulting from the procedure described above using both D- andL-tartaric acid.

Racemic I R Isomer S Isomer C₅₀ (uM) IC₅₀ (uM) IC₅₀ (uM)

<0.05 <0.05 <0.5

Example 10 Induction of Mitotic Arrest in Cell Populations Treated witha Quinazolinone KSP Inhibitor

FACS analysis to determine cell cycle stage by measuring DNA content wasperformed as follows. Skov-3 cells (human ovarian cancer) were split1:10 for plating in 10 cm dishes and grown to subconfluence with RPMI1640 medium containing 5% fetal bovine serum (FBS). The cells were thentreated with either 10 nM paclitaxel, 400 nM quinazolinone 1,200 nMquinazolinone2, or 0.25% DMSO (vehicle for compounds) for 24 hours.Cells were then rinsed off the plates with PBS containing 5 mM EDTA,pelleted, washed once in PBS containing 1% FCS, and then fixed overnightin 85% ethanol at 4° C. Before analysis, the cells were pelleted, washedonce, and stained in a solution of long propidium iodide and 250 μg ofribonuclease (RNAse) A per milliliter at 37° C. for half an hour. Flowcytometry analysis was performed on a Becton-Dickinson FACScan, and datafrom 10,000 cells per sample was analyzed with Modfit software.

The quinazolinone compounds, as well as the known anti-mitotic agentpaclitaxel, caused a shift in the population of cells from a G0/G1 cellcycle stage (2 n DNA content) to a G2/M cell cycle stage (4 n DNAcontent). Other compounds of this class were found to have similareffects.

Monopolar Spindle Formation following Application of a Quinazolinone KSPInhibitor

To determine the nature of the G2/M accumulation, human tumor cell linesSkov-3 (ovarian), HeLa (cervical), and A549 (lung) were plated in96-well plates at densities of 4,000 cells per well (SKOV-3 & HeLa) or8,000 cells per well (A549), allowed to adhere for 24 hours, and treatedwith various concentrations of the quinazolinone compounds for 24 hours.Cells were fixed in 4% formaldehyde and stained with anti-tubulinantibodies (subsequently recognized using fluorescently-labeledsecondary antibody) and Hoechst dye (which stains DNA).

Visual inspection revealed that the quinazolinone compounds caused cellcycle arrest in the prometaphase stage of mitosis. DNA was condensed andspindle formation had initiated, but arrested cells uniformly displayedmonopolar spindles, indicating that there was an inhibition of spindlepole body separation. Microinjection of anti-KSP antibodies also causesmitotic arrest with arrested cells displaying monopolar spindles.

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated withQuinazolinone KSP Inhibitors.

Cells were plated in 96-well plates at densities from 1000-2500cells/well of a 96-well plate (depending on the cell line) and allowedto adhere/grow for 24 hours. They were then treated with variousconcentrations of drug for 48 hours. The time at which compounds areadded is considered To. A tetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (I.S>U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) was usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours was compared to the number of viable cells atthe time of drug addition, allowing for calculation of growthinhibition.

The growth over 48 hours of cells in control wells that had been treatedwith vehicle only (0.25% DMSO) is considered 100% growth and the growthof cells in wells with compounds is compared to this. Quinazolinone KSPinhibitors inhibited cell proliferation in human tumor cell lines of thefollowing tumor types: lung (NCI-H460, A549), breast (MDA-MB-231, MCF-7,MCF-7/ADR-RES), colon (HT29, HCT15), ovarian (SKOV-3, OVCAR-3), leukemia(HL-60(TB), K-562), central nervous system (SF-268), renal (A498),osteosarcoma (U2-OS), and cervical (HeLa). In addition, a mouse tumorline (B16, melanoma) was also growth-inhibited in the presence of thequinazolinone compounds.

A Gi₅₀ was calculated by plotting the concentration of compound in μM vsthe percentage of cell growth of cell growth in treated wells. The Gi₅₀calculated for the compounds is the estimated concentration at whichgrowth is inhibited by 50% compared to control, i.e., the concentrationat which:

100×[(Treated₄ −T ₀)/(Control₄₈ −T ₀)]=50.

All concentrations of compounds are tested in duplicate and controls areaveraged over 12 wells. A very similar 96-well plate layout and Gi₅₀calculation scheme is used by the National Cancer Institute (see Monks,et al., J. NatI. Cancer Inst. 83:757-766 (1991)). However, the method bywhich the National Cancer Institute quantitates cell number does not useMTS, but instead employs alternative methods.

Calculation Of IC₅₀:

Measurement of a composition's IC₅₀ for KSP activity uses an ATPaseassay. The following solutions are used: Solution 1 consists of 3 mMphosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (SigmaA-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (Sigma T-7402), 10ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (Sigma A7906),pyruvate kinase 7 U/ml, L-lactate dehydrogenase 10 U/ml (Sigma P0294),100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (Sigma D9779),5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003-01), and 1 mMEGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions) of thecomposition are made in a 96-well microtiter plate (Coming Costar 3695)using Solution 1. Following serial dilution each well has 50 μl ofSolution 1. The reaction is started by adding 50 μl of solution 2 toeach well. This may be done with a multichannel pipettor either manuallyor with automated liquid handling devices. The microtiter plate is thentransferred to a microplate absorbance reader and multiple absorbancereadings at 340 nm are taken for each well in a kinetic mode. Theobserved rate of change, which is proportional to the ATPase rate, isthen plotted as a function of the compound concentration. For a standardIC₅₀ determination the data acquired is fit by the following fourparameter equation using a nonlinear fitting program (e.g., Grafit 4):

$y = {\frac{Range}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + {Background}}$

Where y is the observed rate and x the compound concentration.

The quinazolinone compounds inhibit growth in a variety of cell lines,including cell lines (MCF-7/ADR-RES, HCT1 5) that express P-glycoprotein(also known as Multi-drug Resistance, or MDR⁺), which conveys resistanceto other chemotherapeutic drugs, such as pacilitaxel. Therefore, thequinazolinones are anti-mitotics that inhibit cell proliferation, andare not subject to resistance by overexpression of MDR⁺ bydrug-resistant tumor lines.

Other compounds of this class were found to inhibit cell proliferation,although GI₅₀ values varied. GI₅₀ values for the quinazolinone compoundstested ranged from 200 nM to greater than the highest concentrationtested. By this we mean that although most of the compounds thatinhibited KSP activity biochemically did inhibit cell proliferation, forsome, at the highest concentration tested (generally about 20 μM, cellgrowth was inhibited less than 50%. Many of the compounds have Gl₅₀values less than 10 μM, and several have GI₅₀ values less than 1 μM.Anti-proliferative compounds that have been successfully applied in theclinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'sthat vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM,doxorubicin is 63 nM, 5-fluorouracil is 1 μM, and hydroxyurea is 500 μM(data provided by National Cancer Institute, Developmental TherapeuticProgram, http://dtp.nci.nih.gov/). Therefore, compounds that inhibitcellular proliferation at virtually any concentration may be useful.However, preferably, compounds will have GI₅₀ values of less than 1 mM.More preferably, compounds will have GI₅₀ values of less than 20 μM.Even more preferably, compounds will have GI₅₀ values of less than 10μM. Further reduction in GI₅₀ values may also be desirable, includingcompounds with GI₅₀ values of less than 1 μM. Some of the quinazolinonecompounds of the invention inhibit cell proliferation with GI₅₀ valuesfrom below 200 nM to below 10 nM.

Example 11

Female nude mice weighing approximately 20 g were implanted s.c. bytrocar with fragments of human tumor carcinomas harvested from s.c.growing tumors in nude mice host. When the tumors were approximately 77mg in size, the animals were pair matched into treatment and controlgroups. Each group containted 8 tumored mice, each of which wasear-tagged and followed individually throughout the experiment. Initialdoses (10 ml/kg of a 66 mM Citrate buffer, pH 5.0/0.9% Saline/10% Tween80 formulation of each test compound having a maximum concentration of 5mg/mL) were given on Day 1 following pair matching, dosing at the levelsand schedules indicated.

Mice were weighed twice weekly, and tumor measurements were taken bycalipers twice weekly, starting on Day 1. These tumor measurements wereconverted to mg tumor weight by a well-known formula, W²×L/2. Theexperiment was terminated when the control group tumor size reached anaverage of 1 gram. Upon termination, the mice were weighted, sacrificedand their tumors excised. Tumors were weighted and the mean treatedtumor weight per group was calculated. In this model, the change in meantreated tumor weight/the change in mean control tumor weight×100%(ΔT/ΔC) was subtracted from 100% to give the tumor growth inhibition(TGI) for each group.

Compounds 1-5 (below) were tested by the above-described method, givingthe results summarized below in Tables A-D. Other compounds of thepresent invention show comparable activities when tested by this method.

TABLE A SKOV3 tumor xenograft Vehicle Compound 1 Taxol Dose & Daily × 580 mg/kg every 20 mg/kg Schedule 3 days × 4 daily × 5 Route i.v. i.v.i.p. # Mice at Start 8 8 8 Final Tumor 904.1 ± 126.5 554.3 ± 76.8 90.4 ±36.0 Weight (Mean ± SEM) Tumor Growth — 41.5% 91.7% Inhibition Mice with0 0 4 Partial Tumor Shrinkage Mean % — — 27.9% Tumor Shrinkage MaximumNone None 16.5% Weight Loss Mortalities 0 1 0

TABLE B SKOV3 tumor xenograft Vehicle Compound 2 Compound 3 Taxol Dose &Daily × 5 50 mg/kg every 60 mg/kg 20 mg/kg Schedule 3 days × 4 daily × 5daily × 5 Route i.v. i.v. i.v. i.p. # Mice at Start 8 8 8 8 Final Tumor1506.3 ± 340.8 ± 93.0 806.1 ± 163.8 55.9 ± Weight 227.1 29.4 (Mean ±SEM) Tumor Growth — 81.5% 48.3% 99.7% Inhibition Mice with 0 0 0 7Partial Tumor Shrinkage Mean % — — — 43.5% Tumor Shrinkage Maximum NoneNone 2.76% 7.49% Weight Loss Mortalities 0 0 1 0

TABLE C SKOV3 tumor xenograft Vehicle Compound 4 Taxol Dose & Daily × 54 mg/kg 20 mg/kg Schedule weekly × 3 daily × 5 Route i.v. i.v. i.p. #Mice at Start 8 8 8 Final Tumor 1191.1 ± 239.6 726.9 ± 147.2 90.6 ± 34.5Weight (Mean ± SEM) Tumor Growth — 40.1% 87.1% Inhibition Mice with 0 04 Partial Tumor Shrinkage Mean % — — 44.4% Tumor Shrinkage Maximum None0.02% 12.26%  Weight Loss Mortalities 1 1 1

TABLE D SKOV3 tumor xenograft Vehicle Compound 5 Taxol Dose & Daily × 525 mg/kg 20 mg/kg Schedule daily × 5 daily × 5 Route i.v. i.v. i.p. #Mice at Start 8 8 8 Final Tumor 1230.4 ± 227.3 405.6 ± 124.8 379.0 ±154.0 Weight (Mean ± SEM) Tumor Growth — 71.0% 73.0% Inhibition Micewith 0 1 0 Partial Tumor Shrinkage Mean % — 56.3% — Tumor ShrinkageMaximum None None 8.77% Weight Loss Mortalities 0 0 0

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1-64. (canceled)
 65. A compound having the formula

R₁ is chosen from alkylaryl and substituted alkylaryl; R₂ is loweralkyl; R₂′ is hydrogen; and the stereogenic center to which R₂ and R₂′are attached is of the R configuration; R₃ is substituted aryl; R₄ issubstituted alkyl; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo or cyano;and R₈ is hydrogen, or a pharmaceutically acceptable salt thereof,wherein substituted alkyl, substituted alkylaryl, and substituted aryl,respectively, refer to alkyl, alkylaryl, or aryl wherein one or morehydrogen atoms are replaced with alkyl, halogen, hydroxy, alkoxy,fluoroalkyl, carboxy, carboalkoxy, carboxyalkyl, carboxamido,sulfonamidoalkyl, sulfonamidoaryl, aminocarbonyl,benzyloxycarbonylamino, cyano,nitro, primary-, secondary- andtertiary-amino, aminoaklylene, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylsulfonamido, arylthio, arylsulfinyl, arylsulfonyl, amidino, aryl,heteroaryl, heterocyclyl, phenoxy, benzyloxy, or heteroaryloxy.
 66. Acompound or salt according to claim 65 wherein R₂ is chosen from ethyl,i-propyl, c-propyl, and t-butyl.
 67. A compound or salt according toclaim 65 wherein R₄ is chosen from lower alkyl, and R₁₆-alkylene-,wherein R₁₆ is amino, lower alkylamino, di(lower alkyl)amino, loweralkoxy, and N-heterocyclyl.
 68. A compound or salt according to claim 65wherein R₇ is halo.
 69. A compound or salt according to claim 65 whereinR₂ is i-propyl; R₃ is methyl- and/or halo-substituted phenyl; R₄ is3-amino-n-propyl; and R₇ is chloro or cyano.
 70. A compound having theformula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is benzyl; R₂is i-propyl; R₂ is hydrogen; R₃ is p-methylphenyl; R₄ is3-amino-n-propyl; R₅ is hydrogen; R₆ is hydrogen; R₇ is cyano; and R₈ ishydrogen.
 71. A compound having the formula

or a pharmaceutically acceptable salt thereof, wherein: R₁ is benzyl; R₂is i-propyl; R₂ is hydrogen; R₃ is p-methylphenyl; R₄ is3-amino-n-propyl; R₅ is hydrogen; R₆ is hydrogen; R₇ is chloro; and R₈is hydrogen.
 72. A method for the treatment or inhibition of restenosisin a subject comprising administering a compound of claim 71 to thesubject.
 73. A method for disrupting mitosis in a cell comprisingcontacting said cell with a compound of claim
 71. 74. The method ofclaim 73 wherein mitotic spindle formation is modulated.